The present invention relates to promoters which permit a caryopsis-specific expression or suppression of genes in genetically modified plants, to methods for the tissue-specific gene expression or gene suppression in plants, expression cassettes, recombinant vectors and host cells containing such promoters, to transgenic plant cells and plants transformed with said promoters, and to methods for generating such plant cells and plants.
Prior-art documents whose disclosure is herewith incorporated into the present application by reference are cited hereinbelow.
The application of plants whose genetic material has been modified with the aid of genetic engineering methods has proved advantageous in many fields of agriculture in order to transfer certain characteristics to crop plants. The predominant aims are firstly crop protection, and secondly improved quality and yield of the plants or products which can be harvested.
A large number of methods for genetically modifying dicotyledonous and monocotyledonous plants are known (cf., inter alia, Gasser and Fraley, Science 244 (1989), 1293-1299; Potrykus, Ann. Rev. Plant Mol. Biol. Plant Physiol. 42 (1991), 205-225; Newell, Mol. Biotechnol. 16(1), (2000), 53-65). They are frequently based on the transfer of gene constructs which, in most cases, constitute combinations of specific coding regions of structural genes with promoter regions and transcription terminators of the same or other (for example heterologous) structural genes.
In connection with the expression of structural genes, providing promoters is of great importance for generating transgenic plants, since the specificity of a promoter is decisive for the point in time at which, the tissue types in which, the physiological conditions under which and the intensity with which a transferred gene is expressed in the modified plant.
To succeed with these various approaches for the genetic manipulation of plants, it is therefore necessary to place genes to be regulated differently under the control of suitable promoters.
Transcriptional initiation and regulation is subject to the DNA segment of a gene termed promoter. As a rule, promoter sequences are in the 5xe2x80x2-flanking region of a transcribed gene. Individual elements of a promoter (for example transcriptional enhancers) can also be located in the 3xe2x80x2-flanking region or within intron sequences (Kuhlemeier, Plant Mol. Biol. 19 (1992): 1-14; Luehrsen, The Maize Handbook, 636-638) (1994).
The controlled expression of transgenes is very useful, for example for introducing resistance properties or the modification of metabolic processes in plants. If a transgene or its gene product is to engage into defined metabolic pathways of a plant, for example if it is to produce a new constituent or to protect from attack by pathogens, its spatially and/or temporarily controlled expression is only possible when an inducible and/or tissue- and/or development-specific promoter is used. Only this makes possible the specific production of desired constituents at a defined developmental stage or within a certain tissue of the plant. For example, when applying antisense technology, where the expression of plant-homologous genes is to be prevented, the use of tissue- and/or development-specific promoters is advantageous over a tissue- and/or developmental-independent expression when, for example, the antisense effect occurs precisely at the developmental stage, or precisely in the tissue, of the plant at which, or in which, the plant-homologous gene is also expressed.
A large number of promoters capable of governing the expression of transferred genes or structural genes in plants is already known. The most frequently used promoter is the 35S CaMV promoter (Franck et al., Cell 1 (1980), 285-294), which leads to constitutive expression of the gene introduced.
Frequently, inducible promoters are also employed, for example for wound induction (DE-A-3843628), chemical induction (Ward et al., Plant Molec. Biol. 22 (1993), 361-366) or light induction (Fluhr et al., Science 232 (1986), 1106-1112).
Under certain circumstances, the use of the frequently described constitutive promoters (e.g. 35 S) entails certain disadvantages. Promoters which bring about a constitutive expression of the genes controlled by them can be employed, for example, for generating herbicide-tolerant and pathogen-resistant plants, but have the disadvantage that the products of the genes controlled by them are present in all parts of the plant, which may be undesirable, for example when the plants are intended for consumption. A negative aspect of tissue- and/or development-independent expression of a transgene can also be an undesired effect on plant development. The use of inducible promoters likewise entails disadvantages, since the induction conditions are typically difficult to control in the open in the case of agricultural plants.
The use of cell- and tissue-specific promoters has also been described: stomata-specific gene expression (DE-A-4207358), seed-, tuber- and fruit-specific gene expression (reviewed in Edwards and Coruzzi, Annu. Rev. Genet. 24 (1990), 275-303; DE-A-3843627), phloem-specific gene expression (Schmxc3xclling et al., Plant Cell 1 (1989), 665-670), root-nodule-specific gene expression (DE-A-3702497) or meristem-specific gene expression (Ito et al., Plant Mol. Biol. 24 (1994), 863-878).
A limited number of promoters which regulate gene expression in the caryopsis are known as yet. The management of certain approaches in the genetic modification of plants therefore requires the provision of alternative promoter systems for gene expression in the caryopsis whose regulation differs from that of the known systems.
Starch biosynthesis genes whose gene products are expressed specifically in the storage tissue of the caryopsis, but not in vegetative tissues, have been isolated from various plant species, for example the relevant genes or cDNA clones of GBSS I. They include the waxy locus from maize (Klxc3x6gen et al. (1986) Mol. Gen. Genet. 203: 237-244), and barley (Rohde et al. (1988) Nucleic Acid Research 16, 14: 7185-7186), rice (Wang et al. (1990) Nucleic Acid Research 18: 5898), potato (van der Leij et al. (1991) Mol. Gen. Genet. 228: 240-248), pea (Dry et al. (1992) Plant J. 2: 193-202), cassava (Salehuzzaman et al. (1993) Plant Mol. Biol. 20: 947-962), millet (Hsingh et al. (1995) EMBL Database Acc.No. U23954) and sugar beet (Schneider et al. (1999) Mol. Gen. Genet. 262: 515-524).
The gene products which are expressed specifically in the caryopsis also include type II starch synthase (SSII). Corresponding genes were isolated from maize (zSSIIa and zSSIIb; Ham et al. (1998) Plant Mol. Biol. 37: 639-649), pea (Dry et al. (1992) Plant J. 2: 193-202), potato (Edwards et al. (1995) Plant J. 8: 283-294) and sweet potato (Ham et al. (1998) Acc. Nr. AF068834).
The situation for wheat is as follows: cDNA clones both for the waxy gene and for the SSII gee were isolated and sequenced. In total, 3 different GBSSI cDNA clones were isolated from wheat (Clark et al. (1991) Plant Mol. Biol. 16: 1099-1101; Ainsworth et al. (1993) Plant Mol. Biol. 22: 67-82 (Block (1997) xe2x80x9cIsolierung, Charakterisierung und Expressionsanalysen von Stxc3xa4rkesynthase-Genen aus Weizenxe2x80x9d [Isolation, characterization and expression analyses of wheat starch synthase genes] (Triticum aestivum L.), PhD thesis, University of Hamburg).
In addition, coding sequences of a type II starch synthase (SSII) have also been isolated from a caryopsis-specific cDNA library, and their caryopsis-specific expression has been detected. Northern analyses have demonstrated that the transcripts of GBSS I (Block (1997), PhD thesis, University of Hamburg, School of Biology) and of SSII (Walter (2000), PhD thesis, University of Hamburg, School of Biology), WO 97/45545, EMBL Database Acc. No. U66377) are found during early developmental stages of the caryopsis, but not in assimilating leaf tissue. In addition, transcripts were also demonstrated in the endosperm and the pericarp for SSII.
Three cDNA sequences of the wheat SSII (T. aestivum L. cv. xe2x80x9cWyunaxe2x80x9d; wSSII-A1, wSSII-B1, wSSII-D1) were furthermore isolated from an endosperm-specific cDNA library (Li et al., (1999) Plant Phys. 120: 1147-1155). Using PCR analyses, each of the three clones was assigned one genome of hexaploid wheat. Western blot analyses have demonstrated that the 100 kDa protein (SGP-B1) is present both in starch-granule-bound foam and in soluble foams during early stages of endosperm development. The isolation and characterization of 3 further SSII cDNA clones (Triticum aestivum L. cv. xe2x80x9cFielderxe2x80x9d, Ss2a-1, Ss2a-2, Ss2a-3) have been described since (Gao and Chibbar (2000) Genome 43: 768-775).
A cDNA clone of a starch-globule-band type II starch synthase (GBSS II) which is expressed not in the endosperm but only in the leaves and the pericarp of wheat has recently been isolated (Vrinten and Nakamura (2000) Plant Physiol.122: 255-263). In diploid wheat (Triticum monococcum L.), a 56 kDa isoform of a GBSS has also been described at the protein level (Fujita and Taira (1998) Planta 207: 125-132). This isoform can be detected in the pericarp, the aleuron and the embryo of immature caryopses.
While three homologous waxy structural genes positioned on chromosomes 7A, 4A and 7D of hexaploid wheat have been isolated in the meantime (Murai et al. (1999) Gene 234: 71-79), the promoter sequences of these or other genomic clones from wheat remain unknown. Only the 5xe2x80x2-flanking regions of GBSS I from barley (GenBank Acc.No. X07931), antirrhinum (GenBank Acc.No. AJ006294), rice (GenBank Acc.No. AB008794, AB008795), potato (GenBank Acc.No. X58453) and maize (GenBank Acc.No. X03935) are known.
If complex tasks in connection with the expression of genes in genetically modified organisms are to be tackled, it is therefore necessary to have a choice between different promoter systems which differ with regard to their specificity. The present invention makes a contribution here.
The aim of the present invention is thus to provide means for making possible a targeted caryopsis-specific gene expression in genetically modified plants, preferably in monocots.
The use of the means according to the invention, i.e. the nucleic acid molecules, vectors, cells or plants according to the invention, makes it possible to engage, in a tissue- and/or development-specifically defined manner, in the plant""s metabolism, for example in the biosynthesis of storage starch, storage fats or storage proteins or else the utilization of the caryopsis as storage or synthesis organ for reserve materials (for example polyglucans, starch, fatty acids, fats, modified or unmodified storage proteins or biopolymers).
Thus, genes can be expressed specifically and at an early point in time in the caryopsis under the control of the nucleic acid molecules or promoter sequences according to the invention, in particular during the grain development of cereals.
Moreover, genes can be suppressed specifically and at an early point of the development in the caryopsis by what are known as xe2x80x9cgene-silencingxe2x80x9d strategies (cosuppression) by means of the promoter sequences according to the invention, in particular during the grain development of cereals. Cosuppression strategies using promoters have been described in detail by Vaucheret et al. (Vaucheret et al., 1998, 16(6), 651-659). The section xe2x80x9cTranscriptional trans-inactivationxe2x80x9d on page 652 of the paper by Vaucheret et al., which specifically describes cosuppression strategies for which the promoters according to the invention are suitable, in particular those which can be termed xe2x80x9cectopic trans-inactivationxe2x80x9d therein (Matzke et al., 1994, Mol. Gen. Genet. 244, 219-229), be herewith incorporated into the present application by reference. Thus, the promoters according to the invention can be used to suppress gene expression of any genes which are under the control of a promoter which is accessible as target for cosuppression by the promoters according to the invention. If appropriate, even a sequence segment of as little as approximately 90 bp in length suffices for this purpose.
The promoters according to the invention thus make possible the targeted modifications of storage starch. Moreover, to make possible the widest possible application of starch for a very wide range of industrial requirements, it is desirable to provide plants which synthesize starches with defined properties. Thus, decisive properties such as solubility, gelatinization behavior, tendency to undergo retrogradation, viscosity and complex formation are determined by the amylose/amylopectin ratio, the degree of branching of the amylopectin and the derivatization of the polymers. A targeted modification of such properties replaces complicated methods for separating amylose and amylopectin or the expensive chemical modification of starch.
A limited possibility of obtaining plants with modified storage starch is the application of traditional plant breeding methods. An (amylose-free) xe2x80x9cwaxyxe2x80x9d wheat was generated successfully by hybridizing spontaneously occurring mutants (Nakamura et al. (1995) Mol. Gen. Genet. 248: 253-259). According to the polyploid character of the commercially important aestivum wheat, mutations relating to the starch structure are not easily recognized since they are compensated for by intact alleles. Thus, the application of traditional plant breeding methods is difficult. Moreover, only enzyme activities which already exist can be resorted to. Novel activities which have hitherto not been identified in plants or which have been identified in plants (or other organisms) which cannot be hybridized with the target plant can also not be improved with the aid of plant breeding methods.
An alternative to traditional plant breeding methods is the targeted modification of starch-producing plants by genetic engineering methods. However, prerequisite herefor is, besides the identification and isolation of genes whose gene products are involved in starch synthesis and/or of starch modification, the use of specific promoters which may be a tissue- and/or development-specific expression of the genes controlled by them in the starch-forming tissues.
Employing the promoter sequences according to the invention also additionally makes possible the integration into the plant genome of genes which impart, to the cereal endosperm, a modified function as storage tissue, for example for storing storage materials other than starches.
These aims are achieved in accordance with the invention by the use forms characterized in the patent claims.
It has been found within the scope of the present invention that a promoter as defined hereinbelow surprisingly brings about, in plants, a caryopsis-specific expression of a coding nucleotide sequence controlled by this promoter.
Thus, the present invention relates to a nucleic acid molecule with the function of a caryopsis-specific promoter, which nucleic acid molecule
a) comprises the nucleic acid sequence defined by the nucleotides 1-4683 of Seq ID No. 1 and corresponding to the one deposited by DSM 14224 (plasmid p. 15/G);
b) comprises one or more sequence elements selected from the group consisting of
i) (Seq ID No. 2);
ii) (Seq ID No. 3);
iii) (Seq ID No. 4);
iv) (Seq ID No. 5);
v) (Seq ID No. 6);
vi) (Seq ID No. 7);
vii) (Seq ID No. 8);
viii) (Seq ID No. 9) and
ix) (Seq ID No. 10);
c) comprises a functional portion of the nucleic acid sequence stated under a);
d) comprises a sequence which hybridizes with at least one of the nucleic acid sequences stated under a) and/or b); and/or
e) comprises a sequence which has at least 60% identity, preferably at least 75% identity, in particular at least 90% identity and very especially preferably at least 95% identity, with one of the nucleic acid sequences stated under a). SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and
The subject matter of the present invention is furthermore a nucleic acid molecule with the function of a caryopsis-specific promoter which
a) comprises one or more sequence elements selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; and
b) comprises a functional portion of Seq ID No.1, preferably one or more sequence elements from the group consisting of nucleotides of positions 1-72, 78-194, 200-402, 409-579, 588-734, 743-837, 852-865, 872-940, 948-957, 962-1113,1120-1180, 1186-1218,1227-1330, 1336-1538,1545-1567, 1574-1589,1597-2015, 2021-2043, 2052-2087, 2096-2276, 2292-2320, 2336-2352, 2361-2470, 2478-2531, 2540-2602, 2609-2712, 2721-2786, 2794-2870, 2883-2937, 2943-2979, 2986-3048, 3056-3073, 3080-3098, 3106-3133, 3142-3155, 3163-3197, 3205-3289, 3302, 3311-3317, 3318-3405, 3414-3446, 3453-3533, 3541-3570, 3578-3617 3617, 3625-3750, 3757-3978, 3988-4031, 4038-4109, 4116-4145, 4153-4173, 4180-4294, 4301-4419, 4427-4449, 4456-4466, 4474-4480, 4489-4683 of Seq. ID No. 1.
The terms xe2x80x9cnucleic acid molecule according to the inventionxe2x80x9d and xe2x80x9cpromoter according to the inventionxe2x80x9d are used synonymously for the purposes of the present invention.
In a preferred embodiment, the promoters according to the invention are those of plant genes, preferably monocots, or derived from such genes. In a further preferred embodiment, the promoters according to the invention are suitable for expressing or suppressing genes in genetically modified organisms, preferably in genetically modified plants, algae or yeasts, especially in genetically modified monocots, and in particular for the expression or suppression of starch synthase genes in said genetically modified organisms. In this context, the promoters according to the invention can be derived from plant genes, obtained from algae or yeasts, modified by recombinant DNA techniques and/or generated synthetically.
The promoters according to the invention can be modified for example by being combined with further cis-regulatory elements. Thus, the promoters according to the invention can additionally be combined with enhancer elements in order to enhance the expression of the corresponding nucleic acid molecule without however influencing its tissue-specific expression. Individual cis-elements (see below) of the isolated promoters can also be combined with each other to give regulatory units.
A measure for the promoter activity is, for example, the expression rate determined for a particular marker gene which is under the regulatory control of the promoter according to the invention. Examples of suitable marker genes are the E. coli xcex2-glucuronidase gene (gus) (Jefferson (1987) Plant Molecular Biology Reporter Vol. 5 (4): 387-405) or the green fluorescence protein gene (gfp) (Baulcombe et al., Plant J. 7 (16) (1993), 1045-1053). The organ or tissue specificity can be determined readily by comparison of the expression rates for said marker genes determined from individual tissues or organs of the plant. Functional portions of the promoter sequence according to the invention comprise, for the purposes of the present invention, naturally occurring variants and also artificial nucleotide sequences, for example those obtained by mutagenesis or chemical synthesis.
In the context of the present invention, a xe2x80x9cpromoterxe2x80x9d is to be understood as meaning a DNA sequence comprising the regulatory portion of a gene, preferably a structural gene. xe2x80x9cRegulatory portionxe2x80x9d of a gene is to be understood as meaning that portion that determines the expression conditions of the gene. The regulatory portion has sequence motifs with which transcriptional factors and RNA polymerase(s) interact and initiate transcription of the coding portion of the gene. In addition, the regulatory portion can comprise one or more positive regulatory elements, known as xe2x80x98enhancersxe2x80x99. Additionally or instead, however, it may also comprise negatively regulatory elements, known as xe2x80x98silencersxe2x80x99. A xe2x80x9cstructural genexe2x80x9d is generally to be understood as meaning a genetic unit of regulatory and coding portions whose gene product is generally a protein. The information for the primary amino acid sequence of the gene product is present in the coding portion of the structural gene, while the regulatory portion determines when, in what tissues, under what physiological conditions and in what quantities the transcript of the coding portion is formed according to whose template the gene product is synthesized.
The term xe2x80x9ccaryopsis-specificxe2x80x9d is to be understood as meaning, for the purposes of the present invention, that a gene under the control of a promoter according to the invention is expressed in the caryopsis, i.e. endosperm, pericarp, aleuron, embryo and/or scutellum, preferably at an early point in time, i.e. earlier than 15 dap (dap=days after pollination), preferably earlier than 10 dap, particularly preferably earlier than 6 dap. For the purposes of the present invention, a lower limit for the expression time of preferably 5 dap, particularly preferably 3 dap, in particular 2 dap and very particularly preferably 1 dap applies for said xe2x80x9cearly expressionxe2x80x9d. In particular, caryopsis specificity for the purposes of the present invention exists when the promoter according to the invention favors the expression of a gene in the caryopsis, preferably in the central starch endosperm, over other tissues such as, for example, mature leaves or roots and brings about a significant increase in the caryopsis, i.e. expression which is increased by a factor of at least 2 to 5, preferably 5 to 10, in particular 10 to 100 or higher.
In the context of the present invention, caryopsis specificity can be analyzed for example by customary reporter gene experiments. To test an isolated promoter sequence for its promoter activity in the caryopsis, the promoter can, for example, be linked operably to a reporter gene, such as, for example, E. coli xcex2-glucuronidase gene in an expression cassette or in a vector for plant transformation. This construct is then used for transforming plants. The expression of xcex2-glucuronidase in the caryopsis is then determined in comparison with other tissues such as, for example, mature leaves or roots, for example as described by Martin et al. (The GUS Reporter System as a Tool to Study Plant Gene Expression, In: GUS Protocols: Using the GUS genes as a Reporter of Gene Expression, Academic Press (1992), 23-43).
The skilled worker is familiar with the term xe2x80x9ccaryopsisxe2x80x9d; it comprises in particular pericarp and endosperm. Since these tissues undergo dynamic development, the development of the endosperm, for example, into various types of cells and tissues correlates with different biochemical activities, owing to differential gene expression. Additional reference may be made to Olsen et al. (Olsen et al., 1999, Trends in Plant Science 4 (7), 253-257).
The promoter according to the invention permits caryopsis-specific gene expression of a coding nucleotide sequence controlled by it. It constitutes an interesting alternative to known endosperm-specific promoters since it is active in the caryopsis at a very early point in time, i.e.  less than 15 dap, preferably  less than 10 dap, in particular  less than 6 dap (dap=days after pollination). The promoter according to the invention allows in particular the expression of genes whose gene products are involved in the starch metabolism of monocots, in particular in wheat, to be governed efficiently.
The promoters according to the invention can moreover be used in many different ways. For example, they make possible the generation of transgenic plants which, owing to a modified metabolism in the caryopsis, show a qualitatively and/or quantitatively modified composition of reserves in their storage tissue, i.e. in the cereal grain.
Besides the promoter which exhibits the entire sequence defined by the nucleotides 1-4683 of Seq ID No. 1 or the sequence deposited accordingly by DSM 14224, the present invention also relates to promoters which exhibit a functional portion of this sequence and which, in plants, bring about a caryopsis-specific expression of a coding nucleotide sequence controlled by them.
A xe2x80x9cfunctional portionxe2x80x9d of the promoter according to the invention is to be understood as meaning, for the purposes of the present invention, those sequences which do not comprise the complete sequence of the promoter, as defined by nucleotide 1-4683 of Seq ID No. 1 or deposited by DSM 14224, but which are truncated. Despite the truncation, a xe2x80x9cfunctional portion of the promoter according to the inventionxe2x80x9d has the caryopsis specificity according to the invention. Sequences comprising a functional portion of the promoter according to the invention of Seq. ID No. 1 preferably exhibit one or more of the segments from Seq ID No. 1 enumerated hereinbelow: 1-72, 78-194, 200-402, 409-579, 588-734, 743-837, 852-865, 872-940, 948-957, 962-1113, 1120-1180, 1186-1218, 1227-1330, 1336-1538, 1545-1567, 1574-1589, 1597-2015, 2021-2043, 2052-2087, 2096-2276, 2292-2320, 2336-2352, 2361-2470, 2478-2531, 2540-2602, 2609-2712, 2721-2786, 2794-2870, 2883-2937, 2943-2979, 2986-3048, 3056-3073, 3080-3098, 3106-3133, 3142-3155, 3163-3197, 3205-3289, 3302, 3311-3317, 3318-3405, 3414-3446, 3453-3533, 3541-3570, 3578-3617, 3625-3750, 3757-3978, 3988-4031, 4038-4109, 4116-4145, 4153-4173, 4180-4294, 4301-4419, 4427-4449, 4456-4466, 4474-4480, 4489-4683. The numbers given indicate the nucleotide positions in Seq. ID No. 1.
A xe2x80x9cfunctional portionxe2x80x9d of the promoter sequence according to the invention is to be understood as meaning in particular also natural or artificial mutations of an originally isolated promoter sequence which have the features according to the invention. The term xe2x80x9cmutationxe2x80x9d encompasses the substitution, addition, deletion, exchange and/or insertion of one or more nucleotides or nucleotide motifs, in particular of cis-elements (see below). The aim of such modifications can be, for example, the generation of fragments, the insertion or repositioning of known nucleotide motifs such as, for example, restriction cleavage sites or cis-elements. Thus, the scope of the present invention also extends for example to those nucleotide sequences which can be obtained by modifying the promoter sequence defined by the nucleotides 1-4683 of Seq ID No. 1 or the promoter sequence deposited by DSM 14224 and which have structural and functional features which are essential according to the invention.
xe2x80x9cFunctional portionsxe2x80x9d of the promoter sequence according to the invention in this context also comprise those promoter variants whose promoter activity is reduced or enhanced compared with the unmodified, that is to say naturally obtainable promoter (wild type).
In particular, a xe2x80x9cfunctional portionxe2x80x9d of the promoter sequences according to the invention are the regions identifiable by deletion analysis (cf. examples part), preferably the sequence segments 2241-4683; 2637-4683; 3569-4683; 4071-4683; 4151-4683 and 4403-4683 of Seq ID No. 1.
In principle, the activity of a eukaryotic RNA polymerase II promoter is caused by the synergistic action of various trans-active factors (DNA-binding molecules such as proteins or hormones) which bind to the various cis-regulatory DNA elements (xe2x80x98cis-elementsxe2x80x99) present in the promoter, generally DNA regions approximately 10-20 nucleotides in length. These factors interact directly or indirectly with individual or several factors of the basic transcription machinery, which eventually leads to the formation of a pre-initiation complex in the vicinity of the transcription start (Drapkin et al., Current Opinion in Cell Biology 5 (1993), 469-476). A module-light construction of the eukaryotic RNA polymerase II promoters can be assumed where the cis-elements (modules), as components of the promoter, specifically determine its activity (Tjian and Maniatis, Cell 77 (1994), 5-8).
Individual subdomains of the promoter according to the invention which potentially mediate tissue specificity can be identified for example by fusion with a minimal-promoter/reporter-gene-cassette. A minimal promoter is to be understood as meaning a DNA sequence comprising a TATA-box located approximately 20 to 30 base pairs upstream of the transcription start, or an initiator sequence (Smale and Baltimore, Cell 57 (1989), 103-113; Zawel and Reinberg, Proc. Natl. Acad. Sci. 44 (1993), 67-108; Conaway and Conaway, Annu. Rev. Biochem 62 (1993), 161-190). Examples of minimal promoters are the xe2x88x9263 to +8 xcex9435S promoter (Frohberg, PhD thesis at the FU Berlin, School of Biology (1994)), the xe2x88x92332 to +14 minimal patatin class I promoter, and the xe2x88x92176 to +4 minimal PetE promoter (Pwee et al., Plant J. 3 (1993), 437-449).
Moreover, subdomains or cis-elements of the promoter according to the invention can also be identified via deletion analyses or mutageneses (Kawagoe et al., Plant J. 5(6) (1994), 885-890). The test for functionality of such a subdomain or cis-elements of the promoter can be effected in planta by detecting reporter gene activity in stably transformed cells.
In a further embodiment, the present invention therefore relates to modifications of Seq. ID No.1 obtained in particular by the di- or multimerization of subdomains or cis-elements of Seq ID No. 1, in particualr the nucleotide sequence 1-4683 of Seq. ID No.1.
In a further embodiment of the invention, an increased promoter activity compared with the wildtype is achieved by combining the promoter according to the invention with one or more xe2x80x98enhancersxe2x80x99.
Various enhancer elements have been described in the literature, all of which generally bring about an increase in the expression in a tissue-specific manner, the tissue specificity generally being determined by the particular enhancer used (Benfey et al., Science 250 (1990), 959-966; Benfey et al., EMBO J. 8 (1989), 2195-2202; Chen et al., EMBO J. 7, (1988), 297-302; Simpson et al., Nature 323 (1986), 551-554).
In addition, there are also enhancer elements such as, for example, the PetE enhancer (Sandhu et al., Plant Mol. Biol. 37 (1998), 885-896), which do not act in a tissue-specific manner and which can therefore be placed before the promoter according to the invention as quantitative enhancer elements in order to increase the expression rate of the controlled gene in the caryopsis without modifying the tissue specificity of the promoter according to the invention.
Furthermore, the synthetic enhancer elements known to the person skilled in the art can also be used; these are, for example, derived from naturally occurring enhancers and/or are obtained by combining enhancer elements.
Likewise, the present invention also relates to promoters which exhibit a nucleotide sequence which hybridizes with the nucleotide sequence defined by the nucleotides 1-4683 of Seq ID No. 1 or deposited by DSM 14224, preferably under stringent conditions, and which promoters exert, in plants, a caryopsis-specific effect on the expression of a coding nucleotide sequence controlled by them.
In this context, the term xe2x80x9cstringent conditionsxe2x80x9d means for example hybridization conditions as they are described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). In particular, stringent hybridization takes place under the following conditions:
Hybridization buffer: 2xc3x97 SSC; 10xc3x97 Denhardt""s solution (Ficoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 xcexcg/ml herring sperm-DNA; 50 xcexcg/ml tRNA; or 0.25 M sodium phosphate buffer pH 7.2, 1 mM EDTA, 7% SDS
Hybridization temperature T=65 to 68xc2x0 C.;
Wash buffer 0.2 xc3x97 SSC; 0.1% SDS;
Wash temperature T=65 to 68xc2x0 C.
Such promoters preferably have a sequence identity of at least 30%, especifically preferably of at least 40%, very preferably of at least 50%, especially preferably of at least 60%, particularly preferably of at least 70%, very particularly preferably of at least 80%, very particularly especially preferably at least 90% and in particular very particularly especially preferably at least 95%, with the nucleotides 1-4683 of Seq ID No. 1 or functional portions according to the invention thereof.
The degree of identity of sequences with the promoter according to the invention can be determined by customary sequence alignment with nucleotides 1-4683 of Seq ID No. 1.
When two sequences to be compared differ in length, the sequence identity preferably refers to the percentage of the nucleotide residues of the shorter sequence, which are identical to the nucleotide residues of the longer sequence. The sequence identity can usually be determined by using computer programs such as, for example, the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). Bestfit exploits the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to determine the segment with the highest sequence identity. When applying Bestfit or another sequence alignment program to determine whether a particular sequence has, for example, 95% identity with a reference sequence of the present invention, the parameters are preferably set in such a way that the percentage identity over the entire length of the reference sequence is calculated and that homology gaps of up to 5% of the total number of nucleotides in the reference sequence are permitted. When using Bestfit, the so-called optional parameters can be left at their default values. The deviations which occur when comparing a given sequence with the above-described sequences of the invention can have been caused for example by addition, deletion, substitution, insertion or recombination. Promoter sequences which, as described above, hybridize with nucleotides 1-4683 of Seq ID No. 1 or the corresponding nucleotides sequence deposited by DSM 14224 are preferably derived from plant organisms, preferably from higher plants, especially preferably from monocots, particularly preferably from Gramineae, very especially from plants of the genus Triticum.
Furthermore, the present invention also relates to promoters which exhibit a functional portion of these sequences and which, in plants, bring about a caryopsis-specific expression of a coding nucleotide sequence controlled by them and which comprise one or more sequences selected from the group comprising the nucleotides 1-4683 of Seq ID No. 1, Seq ID No. 2, Seq ID No. 3, Seq ID No. 3, Seq ID No. 4, Seq ID No. 5, Seq ID No. 6, Seq ID No. 7, Seq ID No. 8, Seq ID No. 9 and Seq ID No. 10.
The present invention furthermore relates to expression cassettes comprising one or more promoters according to the invention. In this context, the term xe2x80x9cexpression cassettexe2x80x9d is to be understood as meaning the combination of a promoter according to the invention with a nucleic acid sequence to be expressed. This nucleic acid sequence can be, for example, a polypeptide-encoding sequence, for example a gene which can be linked to the promoter in sense or antisense orientation. The nucleic acid sequence can also code a nontranslatable RNA, for example an antisense RNA or a ribozyme. These nucleic acid sequences can be used in conjunction with the promoter according to the invention to generate plants with a modified phenotype.
Furthermore, the expression cassettes according to the invention can comprise a transcription termination sequence downstream of the 3xe2x80x2 end of the nucleic acid sequence which is linked to the promoter. In this context, a xe2x80x9ctranscription termination sequencexe2x80x9d is to be understood as meaning a DNA sequence which is located at the 3xe2x80x2 end of a coding gene segment and which is capable of bringing about transcription termination and, if appropriate, the synthesis of a poly-A-tail. An example of such a termination sequence is that of the octopine synthase gene. The skilled worker is familiar with others.
Moreover, the present invention relates to a vector comprising one or more promoters or expression cassettes according to the invention.
In an embodiment which is furthermore preferred, the promoter in the vector according to the invention is linked to restriction cleavage sites or a polylinker, either of which permits integration of any sequences downstream of the promoter. In this context, a xe2x80x9cpolylinkerxe2x80x9d is to be understood as meaning a DNA sequence containing recognition sequences of at least one restriction enzyme, preferably of 5 or more restriction enzymes.
In an especially preferred embodiment, a vector according to the invention additionally comprises a sequence for transcription termination, for example that of the octopine synthase gene, downstream of the promoter or the polylinker.
Thus, the present invention likewise relates to vectors comprising one or more expression cassettes according to the invention. If appropriate, the vectors according to the invention comprise selection markers which are suitable for readily identifying, and, if appropriate, selecting cells comprising the vectors according to the invention following transformation.
In a preferred embodiment, the vectors according to the invention are suitable for transforming plant cells, especially preferably for integrating foreign DNA (for example transgenes) into the plant genome. An example of such vectors are binary vectors, some of which are commercially available.
The present invention furthermore relates to host cells which are genetically modified with a nucleic acid molecule according to the invention, or promoter according to the invention, an expression cassette according to the invention or a vector according to the invention, in particular plant cells or microbial cells, for example of the genus Agrobacterium.
In this context, xe2x80x9cgenetically modifiedxe2x80x9d means that the host cell comprises a promoter according to the invention, an expression cassette according to the invention or a vector according to the invention, preferably stably integrated into the genome of the host cell, and that the promoter, or the expression cassette, has been introduced as foreign DNA into the host cell or beforehand into a precursor of this cell. The host cells according to the invention can therefore be themselves the immediate product of a transformation for the purposes of the present invention or be derived from such cells which comprise a promoter according to the invention or an expression cassette according to the invention. Suitable host cells are prokaryotic cells, in particular bacterial cells, or else eukaryotic cells. Eukaryotic cells can be of plant origin, but also derived from fungi, in particular from the genus Saccharomyces.
In a further embodiment, the invention relates to the use of vectors according to the invention, expression cassettes according to the invention or host cells according to the invention, in particular of the genus Agrobacterium, for transforming plants, plant cells, plant tissues or plant parts.
In an especially preferred embodiment, the host cells according to the invention are plant cells, termed xe2x80x9ctransgenic plant cellsxe2x80x9d hereinbelow.
Furthermore, the present invention also relates to plants comprising plant cells according to the invention. In principle, these plants may belong to any plant species, plant genus, plant family, plant order or plant class which is commercially utilizable. They may be monocots or else dicots. The plants according to the invention are preferably useful plants, i.e. plants which are of agricultural, silvicultural and/or horticultural interest. Preferred in this context are agricultural useful plants, in particular cereal species such as, for example, wheat, oats, barley, rye, maize, rice, fodder and forage grasses (such as, for example alfalfa, white clover or red clover), in particular wheat.
In a further embodiment, the present invention also relates to methods for generating transgenic plant cells and plants, which comprises transforming plant cells, plant tissues, plant parts or protoplasts with a nucleic acid molecule according to the invention, a vector according to the invention, an expression cassette according to the invention or, if appropriate, with a host cell according to the invention, preferably a microorganism, growing the transformed cells, tissues, plant parts or protoplasts in a growth medium, and, when transgenic plants are generated, regenerating plants from these.
In a further embodiment, the invention relates to the use of vectors, expression cassettes or, if appropriate, host cells according to the invention for generating transgenic host cells, in particular transgenic plant cells and plants.
In a further embodiment, the invention relates to a method for the caryopsis-specific gene expression in plants, wherein one or more of the nucleic acid molecules according to the invention is integrated stably into the genome of a plant cell, either directly or by means of one or more of the vectors, expression cassettes or host cells according to the invention, and a plant is regenerated from said plant cell.
In a further embodiment, the invention relates to a method for the caryopsis-specific gene suppression in plants, wherein one or more of the nucleic acid molecules according to the invention is integrated stably into the genome of a plant cell, either directly or by means of one or more of the vectors, expression cassettes or host cells according to the invention, and a plant is regenerated from said plant cell, preferably by means of cosuppression.
The plants according to the invention can be generated by methods known to the skilled worker, for example by transforming plant cells or tissue and regenerating intact plants from the transformed cells or the tissue.
In principle, a multiplicity of molecular-biological techniques is available for introducing DNA into a plant host cell. These techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of the DNA by means of the biolistic approach etc.
When DNA is injected, electroporated and transformed by means of biolistic methods (xe2x80x98particle gunxe2x80x99) into plant cells, no specific requirements as such are made to the plasmids used. Simple plasmids such as, for example, pUC derivatives can be used. However, if intact plants are to be regenerated from cells transformed thus, for example the presence of a selectable marker gene is necessary.
Depending on the method by which desired genes are introduced into the plant cell, further DNA sequences may be required. If, for example, the Ti or Ri plasmid are used for transforming the plant cell, at least the right border, but frequently the right and left border, of the Ti and Ri plasmid T-DNA must be linked to the genes to be introduced as flanking region.
If agrobacteria are used for the transformation, the DNA to be introduced must be cloned into specific plasmids, viz. either into an intermediary vector or into a binary vector. The intermediary vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination owing to sequences which are homologous to sequences in the T-DNA. This Ti or Ri plasmid additionally contains the vir region, which is necessary for transferring the T-DNA. Intermediary vectors are not capable of replication in agrobacteria. The intermediary vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors are capable of replicating both in E.coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187). The agrobacterium acting as the host cell should contain a plasmid carrying a vir region. The vir region is necessary for transferring the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed thus is used to transform plant cells.
The use of T-DNA for transforming plant cells has been studied intensively and described sufficiently in EP 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.
To transfer the DNA into the plant cell, plant explants can expediently be cocultured together with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Then, intact plants can be regenerated from the infected plant material (for example leaf sections, stem segments, roots, but also protoplasts, or plant cells grown in suspension culture) in a suitable medium which may contain antibiotics or biocides for selecting transformed cells. The plants thus obtained can then be examined for the presence of the DNA introduced. Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation have been described (cf., for example, Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pxc3xchler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basle-Cambridge).
Monocots have already been routinely transformed by means of the biolistic approach and by means of agrobacteria (Komari et al., (1998); Advances in cereal gene transfer; Current Opinion in Plant Biotechnology 1, p. 161 et seq.; Bilang et al. (1999), Transformation of Cereals, Genetic Engineering, 12, pp.113-148 Ed.: JK Setlow, Kluwer Academic/Plenum Publisher, New York). Other suitable methods are the electrically or chemically induced DNA uptake into protoplasts, the electroporation of partially permeabilized cells, the macroinjection of DNA into inflorescences, the microinjection of DNA into microspores and proembryos, the DNA uptake by germinating pollen, and the DNA uptake into embryos by soaking (review: Potrykus, Physiol. Plant (1990), 269-273).
The present invention furthermore also relates to the propagation material and harvested material of the plants according to the invention, which comprises plant cells according to the invention.
For the purposes of the present invention, the term xe2x80x9cpropagation materialxe2x80x9d extends to all those constituents of the plant which are suitable for generating progeni via the vegetative or generative route. Examples which are suitable for vegetative propagation are cuttings, callus cultures, rhizomes, root stocks or tubers. Other propagation material encompasses, for example, fruits, seeds, seedlings, protoplasts, cell cultures and the like. The propagation material is preferably tubers or seeds.
The present invention furthermore relates to the use of promoters according to the invention, or to the promoters identified by means of the method according to the invention, for the caryopsis-specific expression of transgenes in plant cells or plants.
Moreover, the present invention relates to the use of the promoters according to the invention, or of the promoters identified by means of the method according to the invention, for the caryopsis-specific cosuppression of genes or transgenes in plant cells or plants.
In this context, the term xe2x80x9ctransgenexe2x80x9d is to be understood as meaning a DNA sequence which has been introduced artificially into a plant and which contains one or more of the nucleic acid molecules according to the invention.
These and other embodiments are disclosed to the skilled worker by the description and the examples of the present invention. Further literature on any of the abovementioned methods, means and applications required for the purposes of the present invention is known to the skilled worker from the prior art. The methods of choice which are suitable for this purpose are, inter alia, public databases (for example xe2x80x9cMedlinexe2x80x9d), some of which are available via the Internet. Other databases and addresses are known to the skilled worker and can be found, if appropriate, on the Internet, for example using any known search engine. An overview over sources and informations on patents or patent applications in biotechnology can be found in Berks, TIBTECH 12 (1994), 352-364.
To describe the invention in greater detail, one of the promoters is represented by Seq ID No.1, consisting of 4 683 bases of the genomic sequence of the isolated SSII subclone p15/G such as deposited by DSM 14224.
Seq ID No.1 represents the DNA sequence of the genomic SSII subclone p15/G (insert size: 5057 base pairs). Nucleotides 1-4683 of Seq. ID No. 1 correspond to the 5xe2x80x2-flanking region of the gene, i.e. the SSII promoter. Subclone p15/G additionally contains 374 bases of the SSII structural gene (positions 4684 to 5057). An intron with a length of 91 bases is present at positions 4948 to 5038.
The isolated SSII cDNA clone (Walter (2000), PhD thesis University of Hamburg, School of Biology, WO 97/45545, or EMBL Database U66377) shows 83.7% homology with the genomic sequence stated in Seq ID No.1 in the 5xe2x80x2-untranslated region (positions 4512 to 4683). In the first exon (positions 4684-4947), there is 92.6% sequence identity with this cDNA sequence. The 5xe2x80x2-untranslated region and the first exon of the sequence stated under Seq ID No.1 (positions 4596-4947) is identical to the first 352 bp of a CDNA clone of an SSII (Theor. Appl. Genet., (1999), 98: 1208-1216), WSSIIA and the first 318 bp of the cDNA clone wss2a-2 (AJ269503), which has been published by Gao et al. (2000, loc.cit.).
The 5xe2x80x2-flanking DNA region of the isolated genomic clone (Seq ID. No.1, i.e. nucleotides 1-4683 of Seq. ID No. 1) has been compared with sequences which have already been published by means of database searches. The promoter region of a wheat SSII gene is a previously unknown sequence.
Moreover, the described DNA sequence, which flanks the start codon 5xe2x80x2 (Seq ID No.1, nucleotides 1-4683) was searched in the PLACE database (http://www.dna.affrc.go.jp/htdocs/PLACE/; Web Signal Scan Program) for DNA motifs with sequence homology with known cis-regulatory elements. In the SSII promoter (i.e. nucleotides 1-4683 of Seq. ID No. 1, as deposited by DSM 14224), the following endospermxe2x80x94or seed-specific cis-regulatory DNA elements were identified:
Sequence homologies with elements which participate in gene expression which is regulated by sugar were found at the following positions:
Sequence homologies with DNA elements which participate in hormonally-regulated gene expression by ABA or GA were found at the following positions:
Sequence homologies with elements which participate in a hormonally regulated gene expression by auxin or ethylene were found at the following positions:
Sequence homologies with DNA elements which represent a light- or temperature-regulated gene expression were found at the following positions:
In addition to the sequence motifs described, homologies with DNA motifs for general transcription factors (for example GT1 consensus, G boxes, DOF boxes, GATA motifs, Myb and Myc boxes; for information, see PLACE database), and also T boxes and ARS elements (Gasser S. M. et al. (1989) Intnatl Rev Cyto 119:57-96) were found in the promoter.
The SSII promoter stated under Seq ID No.1 also exhibits sequence motifs which have not been described as yet. They include a motif of the sequence 5xe2x80x2-AAAAATGT-3xe2x80x2, which occurs in total nine times in the region of from 3009 to 3329 of the sequence stated under Seq ID No.1 (positions: 3009, 3030, 3114, 3157, 3177, 3199, 3275, 3307, 3321). In contrast to this motif, the xe2x88x92300 element, also termed prolamin box, has the sequence motif 5xe2x80x2-TGTAAAG-3xe2x80x2 and is located approximately 300 nucleotides from the transcription start in promoters of hordeins (barley), gliadins and LMW glutenins (wheat) and also xcex1-zeins (maize) (Forde et al. (1985) Nucleic Acid Research 13: 7327-7339; Mena et al. (1998) Plant J. 16: 53-62).
Repeating short sequence motifs are located at position 4221 (TCTA)4, at position 2304 (GCCT)3 and position 2364 (GCT)3. A direct repeat of sequence AAAAATGTAATCAAGCTTT (SEQ ID NO: 17) is located at positions 3199 and 3275. In the 5xe2x80x2-untranslated region directly before the translation start (position 4671 in Seq ID NO. 1) there is a GC-rich sequence CCCGGCCGCC (SEQ ID NO: 18), which is also present in the 5xe2x80x2-untranslated region of the maize zSSIIa cDNA clone before the translation start (Genbank Acc. No. AF019296; Harn et al. (1998) Plant Mol. Biol. 37: 639-649).
The genomic SSII subcdone p8/C, which is disclosed in German Patent Application DE10032379.0 and deposited by DSM 13397 constitutes a fragment of the sequence ID No. 1. In this respect, the content of DE10032379.0 is expressly incorporated into the present application by reference.
Deposition of Microorganisms
The nucleic acid molecule according to the invention as shown in Seq ID No. 1 was deposited at the Deutsche Sammiung fxc3xcr Mikroorganismen und Zelikulturen (DSMZ) in Brunswick, Germany, in compliance with the provisions of the Budapest Treaty by means of plasmid DNA:
On Apr. 6, 2001, plasmid p15/G comprising Seq ID No.1 was deposited at the DSMZ under deposition number DSM 13398.
Cloning Methods
The vectors pBluescript(trademark) II, SK(+/xe2x88x92) and KS(+/xe2x88x92) phagemid vectors (Stratagene GmbH, Heidelberg, Germany) and Lambda Fix(copyright) II/Xhol cloning vector (Stratagene GmbH, Heidelberg, Germany) were used for cloning into E.coli bacterial strains.
Bacterial Strains
The E.coli strains DH5xcex1 (Life Technologies, Eggenstein, Germany) and Epicurian Coli SURE(copyright) (Stratagene GmbH, Heidelberg, Germany) were used for the Bluescript vectors. The Epicurian Coli strain XL1-Blue MRA (Stratagene) was used for the bacteriophage vectors.
As regards basic techniques in molecular biology or, for example, buffer compositions, reference is made to Sambrook et al. ((1989), Molecular Cloning; A Laboratory Manual, Second Edition; Cold Spring Harbour Laboratory Press).
The examples which follow illustrate the invention, but do not limit it in any way whatsoever.